Two coordinated signals are required to elicit productive humoral immune responses from nave follicular B cells. The first is provided immediately at the cell surface when the B cell antigen receptor (BCR) recognizes polyvalent or membrane-restricted antigens and initiates an interrelated web of signaling cascades. In contrast, the necessary second signals arise later from processes exclusive to specialized late endosomes (the MHC II containing compartment or MIIC). These include the loading of MHC class II with peptides, and subsequent recruitment of T cell help, and ligand binding by toll-like receptors (TLR) 7 and 9. Activation of these signal 2 enabling receptors is dependent upon the abilities of the BCR to capture ligands at the cell surface and to deliver them along the endocytic pathway to their receptors. However, the molecular mechanisms by which the BCR target late endosomes, and thereby couples signal 1 with signal 2, are poorly understood. We have previously demonstrated that Ig ubiquitination is necessary in cell lines for targeting internalized BCR complexes to late endosomes. We have also demonstrated that BCR endocytic targeting to the MIIC is not a constitutive process but one that is regulated in peripheral anergic cells to prevent aberrant B cell activation. We have now gone on to derive an in vivo model of Ig ubiquitination (Ig???) that confirms its role in ushering BCR complexes into late endosomes. In addition, we now demonstrate that Ig ubiquitination is required for selection of IgMhigh cells into the immature B cell pool and for both Td and Ti humoral immune responses. The requirement for Ig ubiquitination was independent of antigen specificity and was associated with specific defects in basal and BCR-induced signaling. Complementation of these signaling defects in vitro restored BCR targeting to late endosomes. Based on these and other data presented in Preliminary Results, we hypothesize that BCR ubiquitination, much like tyrosine phosphorylation, plays a critical role in coupling the BCR to signaling processes necessary for B cell development and peripheral activation. Furthermore, we propose that ubiquitination plays additional roles in BCR homeostasis and in selecting and maintaining B cells irrespective of antigenic specificity. This central hypothesis will be tested in the followin Specific Aims: Aim 1. To determine how Ig ubiquitination contributes to immature B cell selection. Aim 2. To determine the role of Ig ubiquitination in peripheral immune responses. Aim 3. Determine the mechanisms by which Ig regulates BCR signal initiation and surface homeostasis. !